PROJECT SUMMARY Billions of people are at risk of contracting vector-borne diseases. Dengue alone causes 90 million infections per year globally and like many vector-borne diseases, currently there are no drugs or vaccines to treat or prevent these infections. Therefore, vector control is the primary tool used for vector-borne disease prevention. In recent years, novel vector population suppression technologies have been created (e.g. RIDL and Wolbachia based systems), but production of mosquitoes for these programs is labor intensive and is limited in scalability and distribution. In this study, we will use a functional genomic screening approach to identify key sex determinate, female essential (FE) and male fertility (MF) genes in the dengue vector, Ae. aegypti. These studies will improve our understanding of the biology of this important vector and it can be used to inform the design of new genetic population suppression methods to control this vector. After these genes are identified and characterized, we will incorporate them into the design of precision guided sterile insect technique (pgSIT) technologies in an attempt to overcome limitations in traditional SIT control strategies. Sterile insect technique (SIT) is the gold standard for insect population control but has many limitations. Our proposed technology aims to simultaneously knock-out FE and MF genes using a binary CRISPR/Cas9 system in the Ae. aegypti disease vector. One line will target one or more female essential FE genes and one or more MF genes and the other line will express a Cas9. When these two lines are crossed, they create sterile, male progeny that are ready for release into a population suppression program. To generate these lines, initially we will characterize >40 candidate FE and MF genes A. aegypti in single and combinatorial sgRNA screening assays in our previously characterized Cas9 expression. These genes will be initially selected through transcriptomics, comparative genomics and functional genomic studies. Gene targets that exhibit consistent FE or MF phenotypes will then be engineered into transgenic Ae. aegypti line expressing guide RNAs (gRNA) targeting these genes. These lines will then be crossed to multiple Cas9 lines and the fitness of each line and their F1 progeny will be determined over many generations to ensure population stability. The design and integration of these transgenes will then be varied and optimized to facilitate improved, stable and consistent phenotypes. These optimization experiments will also address multiple fundamental questions about lethal biallelic mosaicism, a phenomenon identified as driving pgSIT success in D. melanogaster, and endogenous Cas9 expression systems, including the impact of transgene expression timing and transgene location on the long-term stability of the lines. The optimal design and genes will then be evaluated in fitness and small population cage studies. In the end, we aim to identify novel FE and MF genes that will allow us to better understand mosquito biology and which allow us to create a genetic SIT system that improves upon traditional SIT technologies.